Specnuezhenide suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis

Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM) and is associated with osteoclast formation and enhanced bone resorption. Specnuezhenide (SPN) is an active compound with anti-inflammatory and immunomodulatory properties. However, the roles of SPN in diabetic osteoporosis remain unknown. In this study, primary bone marrow macrophages (BMMs) were pretreated with SPN and were stimulated with receptor activator of nuclear factor kappa B ligand (RANKL; 50 ng/mL) to induce osteoclastogenesis. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of cellular oncogene fos/nuclear factor of activated T cells c1 (c-Fos/NFATc1), nuclear factor kappa-B (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis. NF-κB luciferase assays were used to examine the role of SPN in NF-κB activation. The DM model group received a high-glucose, high-fat diet and was then intraperitoneally injected with streptozotocin (STZ). Micro-CT scanning, serum biochemical analysis, histological analysis were used to assess bone loss. We found that SPN suppressed RANKL-induced osteoclast formation and that SPN inhibited the expression of osteoclast-related genes and c-Fos/ NFATc1. SPN inhibited RANKL-induced activation of NF-κB and MAPKs. In vivo experiments revealed that SPN suppressed diabetes-induced bone loss and the number of osteoclasts. Furthermore, SPN decreased the levels of bone turnover markers and increased the levels of runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), calcium (Ca) and phosphorus (P). SPN also regulated diabetes-related markers. This study suggests that SPN suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis, and provides an experimental basis for the treatment of diabetic osteoporosis.


Introduction
Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM), and is generally caused by an absolute or relative deficiency of insulin, a hormonal imbalance caused by endocrine dysfunction, or calcium (Ca) and phosphorus (P) metabolism disorders, resulting in decreased bone density and changes in bone microstructure [1]. Recently, an increasing number of studies have noted that diabetes has a negative impact on bone, and the risk of fracture in patients can be up to 1-3 times more likely. Osteoporosis is characterized by decreased bone mass and bone strength [2][3][4][5]. The major pathological feature of osteoporosis is the increased osteoclast formation and activation [6]. Pathologically over-activated osteoclasts promote bone resorption and inhibit bone formation by inhibiting the activity of osteoblasts, leading to progressive bone loss in osteo-porosis [7,8]. Therefore, osteoclasts are the main target of drugs for the treatment of osteoporosis.
As a classic inducer of osteoclast formation, receptor activator of nuclear factor kappa B ligand (RANKL) plays a decisive role in osteoclast formation, activation and survival [9,10]. The interaction between RANKL and its receptor RANK in osteoclast precursor cells triggers a series of signaling pathways, including mitogen-activated protein kinases (MAPKs), nuclear factor kappa-B (NF-κB), activator protein 1 (AP-1), and nuclear factor of activated T cells c1 (NFATc1) [11][12][13], among which the NF-κB pathway and MAPK pathway are the two most important signaling pathways in the process of osteoclast formation [14,15]. Studies have shown that mice with MAPK-related gene deletion can present with osteoclast formation disorders and a higher risk of osteosclerosis [16,17]. In addition, the NF-κB signaling pathway is also an important research target for the treatment of osteoporosis, and molecules in the pathway, including protein phosphorylation and nuclear translocation, may become drug targets [18,19].
In recent years, there have been an increasing number of studies on the key signaling pathways and cytokines extracted from traditional Chinese medicine to regulate the formation and function of osteoclasts, which has also brought new options for the treatment of osteoporosis [20]. Specnuezhenide (SPN; C 31 H 42 O 17 , molecular weight 686.62; Figure 1A) is an active constituent of soluble fissured cycloiridoid glycosides in Ligustrum [21]. Studies have shown that SPN has inhibitory effects on MAPK/extracellular regulated protein kinases (ERK), hypoxia-inducible factor 1-alpha/vascular endothelial growth factor (HIF-1α/VEGF) and other inflammation-related signaling pathways and has anti-inflammatory, immunomodulatory and anti-aging properties [22][23][24]. Due to the recent discovery of SPN, there are few studies on it, and its medicinal value is not clear [23]. The anti-inflammatory and immunomodulatory properties of SPN may justify its role in osteolytic bone disease. However, to date, the direct effect of SPN on diabetesinduced osteoporosis has not been investigated.
Streptozotocin (STZ) is an antibiotic extracted from Streptomyces sp [25]. STZ can specifically destroy islet β cells, causing autoimmune inflammatory damage and apoptosis of islets [26]. Damage to islet β cells leads to a rapid decrease in insulin secretion and a significant increase in blood sugar, showing the appearance of diabetes [26]. Numerous studies have established a rat model of diabetic osteoporosis by combining a high-glucose, high-fat diet with streptozotocin therapy [27,28]. In this study, we investigated the effect of SPN on osteoclast formation and diabetic osteoporosis, which provided a research basis for the possibility of natural compounds as alternative drugs for the prevention and treatment of osteoporosis.

Materials and Methods Chemicals
SPN (purity>98%) was obtained from Shanghai Pureone Biotechnology (Shanghai, China). A luciferase assay system was obtained from Promega (Madison, USA). RANKL was obtained from Sigma (St Louis, USA). Primary antibodies and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Fetal bovine serum (FBS) and alpha-modified Eagle's medium (α-MEM) were purchased from Gibco BRL (Grand Island, USA). Recombinant macrophage-colony stimulating factor (M-CSF) and RANKL were obtained from R&D Systems (Minneapolis, USA). Other chemicals were purchased from Sigma.

Osteoclastogenesis in vitro
Primary bone marrow macrophages (BMMs) were isolated from the femur and tibia of male C57BL/6 mice (6-8 weeks old) as described previously [29]. BMMs were cultured in α-MEM supplemented with 10% FBS, 1% antibiotics (100 U/mL penicillin and streptomycin) and 20 ng/mL M-CSF at 37°C with 5% CO 2 . To explore the role of SPN in BMM osteoclastogenesis, BMMs were plated into 96-well plates (0.32 cm 2 ) at 1×10 4 cells/well. Then, BMMs were pretreated with various concentrations of SPN (0, 10, 50, and 200 μM) for 30 min and cultured with 50 ng/mL RANKL for 5 days. The medium was replaced every 2 days. After 5 days, the cells were fixed with 4% paraformaldehyde, and tartrate-resistant acid phosphatase (TRAP) staining was performed at 37°C. TRAP-positive cells containing ≥3 nuclei were considered osteoclasts and counted using ImageJ software (National Institutes of Health, Bethesda, USA).

RNA extraction and quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was used to measure the expressions of specific genes during osteoclast formation. To investigate whether SPN regulates the expression of osteoclast-associated genes in a dose-dependent manner, BMMs were pretreated with SPN at concentrations of 0, 50, and 200 μM for 30 min and stimulated with RANKL for 5 days. In addition, to investigate whether SPN regulated the expressions of osteoclast-associated genes in a time-dependent manner, BMMs were pretreated with 200 μM SPN for 30 min and stimulated with RANKL for 0, 1, 3, and 5 days. Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Total RNA was used for reverse transcription to synthesize cDNA with a cDNA Synthesis Kit (Thermo Fischer Scientific, Waltham, USA). qRT-PCR was performed using SYBR Premix Ex Taq II (Takara, Tokyo, Japan) with an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, USA). β-Actin was used as an internal control for mRNA. The primers used in this study are listed in Table 1.

Establishment of the diabetes osteoporosis rat model
Eight-week-old male Sprague-Dawley (SD) rats were obtained from GemPharmatech Co., Ltd (Nanjing, China). A total of 24 SD rats were randomly divided into 4 groups: the control group; the DM model group; the DM model+50 μM SPN (0.035 mg/kg) group; and the DM model+200 μM SPN (0.14 mg/kg) group. The DM model group received a high-glucose, high-fat diet for five weeks, and was then intraperitoneally injected with STZ (60 mg/kg). After one week, a diagnosis of diabetes was made based on blood glucose (fasting) >11.1 mM. For the SPN-treatment group, 50 μL of SPN solution dissolved in saline (50 μM, 0.035 mg/kg) or 200 μL of SPN solution dissolved in saline (200 μM, 0.14 mg/kg) were injected intraarticularly into DM model rats every 7 days. After 6 weeks of treatment, the rats were sacrificed, and their femurs were collected for follow-up experiments. This in vivo experiment was approved by the Affiliated Geriatric Hospital of Nanjing Medical University [Permit number SYXK(SU)2018-0020].

Micro-CT scanning
The fixed femurs were scanned using high-resolution microcomputed tomography analysis (70 kVp, 110 μA, and 9 μm; Bruker, Kontich, Belgium). Three-dimensional (3D) reconstructions of the femoral structure were performed after scanning. The region of interest (ROI) was generated from the region 5 mm above the growth plate on the distal femur with a height of 1 mm. Then, a constant trabecular binarization threshold was used to assess the bone parameters in the ROI, bone volume/total volume (BV/TV; %), trabecular separation (Tb.Sp, 1/mm), trabecular thickness (Tb. Th, 1/μm), and trabecular number (Tb.N, 1/μm) were used to assess bone loss using CT Analyzer software.

Serum biochemical analysis
After 6 weeks of treatment, the rats were sacrificed by excessive isoflurane. Blood was collected and centrifuged for 15 min to isolate

Histological analysis
For histological analysis, the metaphyseal region of femoral bones was fixed in 4% paraformaldehyde for 1 day. After decalcification in 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks, the sections were embedded in paraffin, sectioned at 5 μm thickness and stained with hematoxylin and eosin (H&E).

Statistical analysis
All results are presented as the mean±standard deviation (SD) of three experiments. Statistical significance was determined using SPSS software (ver. 22). Significant differences were analyzed by one-way ANOVA or two-tailed Student's t-test. P<0.05 was considered statistically significant.

Specnuezhenide inhibits RANKL-induced osteoclastogenesis
To explore the role of SPN in RANKL-induced osteoclastogenesis, functional experiments on BMMs are listed in Supplementary Figure  S1. To determine whether SPN has toxic effects on BMMs, MTT assay was used to evaluate cell viability. The results showed that even with SPN at 200 μM for 48 h, the cells still maintained a high survival rate ( Figure 1B). However, 400 μM SPN had a slightly toxic effect on BMMs ( Figure 1B). We thus selected 200 μM as the SPN concentration for subsequent experiments. The effects of SPN on RANKL-induced osteoclast formation were first examined. BMMs were pretreated with different concentrations of SPN (0, 10, 50, and 200 μM) and incubated with RANKL for 3 days. After 3 days, the cells were stained with TRAP ( Figure 1C). TRAP-positive cells with more than three nuclei were considered osteoclasts. The results showed that SPN dose-dependently inhibited RANKL-induced osteoclast formation ( Figure 1D). Therefore, we conclude that the inhibition of osteoclast formation by SPN is not induced by its toxicity to mature osteoclasts.

Specnuezhenide suppresses RANKL-induced gene expression
nuclear factor of activated T cells 1 (NFATc1) is a key regulator of late osteoclast differentiation and is required for the regulation of many osteoclast-specific genes [33,34]. TRAP, Cathepsin K (CTSK), matrix metalloproteinase-9 (MMP-9), dendritic cell-specific transmembrane protein (DC-STAMP), c-FOS and NFATc1 play important roles in the dissolution of bone matrix [35,36]. Therefore, we used qRT-PCR to examine the effect of SPN on the expressions of osteoclast-associated genes, including TRAP, CTSK, MMP-9, DC-STAMP, c-FOS and NFATc1. To investigate whether SPN regulates the expressions of osteoclast-associated genes in a dose-dependent manner, BMMs were pretreated with SPN at concentrations of 0, 50, and 200 μM for 30 min and stimulated with RANKL for 3 days. As shown in Figure 2, SPN inhibited the expressions of all marker genes in osteoclasts in a dose-dependent manner, consistent with its inhibitory effect on osteoclast formation. These results indicate that SPN suppresses RANKL-induced osteoclast-associated gene expression.

Specnuezhenide suppresses RANKL-mediated expression of c-Fos/NFATc1
NFATc1 and c-Fos are two crucial transcription factors of osteoclast differentiation that are induced and activated by the RANKL/RANK signaling pathway [37,38]. Therefore, the effect of SPN on NFATc1 and c-Fos protein expression was detected by western blot analysis. BMMs were pretreated with 200 μM SPN for 30 min and stimulated with RANKL for 0, 1, and 3 days. As shown in Figure 3, the protein expressions of NFATc1 and c-Fos were significantly increased during osteoclast generation (1-3 days), whereas 200 μM SPN significantly inhibited their expressions, consistent with previous mRNA levels. Taken together, these results demonstrate that SPN inhibits osteoclastogenesis by downregulating the expressions of c-Fos and NFATc1.

Specnuezhenide suppresses RANKL-induced NF-κB activation
The activation of NF-κB induced by RANKL is crucial for osteoclast biology [37]. Therefore, NF-κB transcriptional activity was determined. BMMs were transfected with an NF-κB luciferase reporter construct and pretreated with SPN (0, 50, and 200 μM) for 30 min, followed by stimulation with 50 ng/mL RANKL for 6 h. The NF-κB luciferase reporter assay results showed that only treatment with RANKL increased the luciferase activity, and SPN significantly inhibited RANKL-induced NF-κB activity in a dose-dependent manner ( Figure 4A). Then, to explore the role of SPN on RANKL-mediated IκB-α expression, BMMs were pretreated with SPN (200 μM) for 30 min, and then treated with 50 ng/mL RANKL for different time (0 to 60 min). As shown in Figure 4B,C, SPN strongly suppresses IκB-α degradation, with the most significant reduction at 20 min.

Specnuezhenide suppresses RANKL-induced MAPK activation
RANKL can activate MAPK pathways during osteoclast formation [39]. To further explore the molecular mechanism of SPN inhibition of c-Fos and NFATc1 expression and activation of NF-κB, we observed the effects of SPN on RANKL-induced MAPK pathways. As shown in Figure 5A,B, RANKL stimulation led to peaks in the levels of p-p38/p38, p-JNK/JNK, and p-ERK/ERK. SPN significantly attenuated the levels of p-p38/p38, p-JNK/JNK, and p-ERK/ERK after 5 min, respectively ( Figure 5A,B). These results indicate that the mechanism by which SPN inhibits osteoclastogenesis involves the inhibition of the MAPK (ERK, p38, and JNK) signaling pathway.

Specnuezhenide suppresses diabetes-induced bone loss
Finally, we tested the effect of SPN in DM animal model. In the animal experiment, DM model rats were injected with SPN at doses of 50 μM or 200 μM. Serum was isolated from each rat for biochemical analysis. Compared with the control group, the model 1083 Specnuezhenide suppresses diabetes-induced bone loss group showed increased levels of blood glucose, HbA1c, LDL-C, TC, and TG, and decreased levels of insulin and HDL-c, while the SPNtreated group exhibited opposite effects on these diabetes-related markers ( Table 2 and Table 3). In addition, 200 μM SPN treatment decreased serum bone turnover markers, such as osteocalcin, ALP, TRACP 5b, PINP, and CTX-1 (Table 4). SPN also decreased RANKL level and increased RUNX2, OPG, Ca, and P levels ( Table 5). Micro-CT analysis showed that 200 μM SPN suppressed bone mass loss in the DM model, while 50 μM SPN had no effect on bone mass loss ( Figure 6A). Quantitative analysis confirmed that the 200 μM SPNtreatment group had increased bone parameters (BV/TV, Tb.N, Tb. Th), and decreased Tb.Sp compared with the model group ( Figure  6B). H&E staining further confirmed that diabetes-induced bone mass loss was significantly reduced in the 200 μM SPN-treatment group compared with the model group ( Figure 6C). These results indicate that SPN suppresses diabetes-induced bone loss.

Discussion
With the increasing improvement of living standards, DM has become the most common endocrine and metabolic disorder threatening human health, accompanied by the occurrence of serious complications [40]. Among these disorders, diabetic osteoporosis has become a hot topic of discussion [41]. Diabetic osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and degeneration of bone microstructure [42]. Strotmeyer et al. [13] found that among patients with diabetes, approximately 50%-66% showed a decreasing trend in bone mineral density, and approximately 33% were diagnosed with osteoporosis. Moreover, studies revealed that compared with that in healthy individuals, the risk of fracture in diabetic patients is increased by 2-3 times, and the risk increases over time; when diabetes lasts for more than 15 years, the risk is 3 times greater than that in the healthy individuals [14]. Therefore, diabetic osteoporosis has become a particularly promi-

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Specnuezhenide suppresses diabetes-induced bone loss nent worldwide topic seriously affecting human health. However, its pathogenesis is still unclear, affecting its effective prevention and treatment.
In recent years, natural compounds extracted from plants have attracted increasing attention as anti-bone resorption drugs. A series of natural compounds, such as luteoloside [43], aliseol-B [44], andrographolide [45], (+)-vitisin A [46], matairesinol [47] and quercetin [48], reportedly play an important role in the treatment of osteoclast-associated osteolytic diseases. Therefore, natural compounds may be a new treatment scheme for osteoclast-associated   1085 Specnuezhenide suppresses diabetes-induced bone loss osteolytic diseases, and the study of the mechanism and application of natural compounds on the biological effects of osteoclasts is worthy of attention and further study. SPN was isolated from the fruit of Ligustrum Ligustri, and studies have shown that it plays an important role in the neural system and diabetic retinopathy [49,50]. Ma et al. [51] found that SPN could inhibit chondrocyte inflammation by inhibiting the transmission of NF-κB and Wnt/βcatenin signals, thus playing an anti-inflammatory role in osteoarthritis. The role and regulatory mechanism of SPN in diabetic osteoporosis disease are still unclear. In the present study, we clarified the positive role of SPN in osteoclast formation. Osteoclasts are large multinucleated cells formed by macrophages that are responsible for bone absorption and release of mineral matrix and play an important role in the pathological destruction of bone. In this study, we investigated the effect of SPN on diabetic osteoporosis and its regulatory mechanism. First, the biological function of SPN was evaluated by osteoclast differentiation assay, and the results showed that SPN had an inhibitory effect on osteoclast formation in vitro. More importantly, the MTT assay showed that SPN inhibited osteoclast activity but did not affect cell activity at concentrations up to 200 μM. This result suggests that our study provides a relatively safe treatment option for osteoclast-associated diseases, as SPN does not cause nonspecific cytotoxicity or result in adverse side effects. Then, we detected the expression levels of osteoclast differentiation-related genes, consistent with the inhibitory effect of osteoclasts, and observed reduced expressions of osteoclast marker genes, such as cathepsin K, TRAP, and calcitonin receptor, indicating that the inhibitory effect of SPN on osteoclast formation is dose-dependent.
In addition, two key factors in osteoclast formation have been reported, i.e., c-Fos and NFATc1, which promote the expressions of downstream genes [52,53]. c-Fos induces NFATc1 to modulate osteoclast formation after RANKL stimulation [54]. Consistent with previous results, we found that SPN significantly reduced NFATc1 and c-Fos protein expressions during osteoclast differentiation by western blot analysis. RANKL-induced upregulation of c-Fos and NFATc1 mRNA and protein was dramatically downregulated by SPN pretreatment, indicating that the c-Fos/NFATc1 pathway is a target of the inhibitory effect of SPN on osteoclast differentiation. The inhibitory effect of SPN on the expressions of osteoclast-specific genes suggests that SPN may inhibit some signaling pathways of osteoclast differentiation.
The combination of NFATc1 and other transcription factors further activates osteoclast-specific genes, further explaining the previous assayed results. RANKL binding to its receptor (RANK) on osteoclast precursors leads to a cascade of intracellular events, including NF-κB, AKT, MAPKs, NFAT, ionic calcium and calcium/ calmodulin-dependent kinase. Among these signaling pathways, NF-κB and MAPKs are two major pathways related to osteoclastogenesis [55,56]. RANKL-induced NF-κB activation occurs early in osteoclast differentiation and induces the expressions of osteoclastassociated genes [57,58]. RANKL binding causes the binding of its receptor RANK to TRAF6, forming a complex to activate downstream TAKl and induce IKKα [59]. Activated IKKα regulates the degradation of IκBα. NF-κB is released through the degradation of IκBα and then translocates from the cytoplasm to the nucleus, and subsequently activates osteoclastogenesis gene transcription directly to modulate osteoclast formation and function [60][61][62]. Zhou Table 3

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Specnuezhenide suppresses diabetes-induced bone loss et al. [61] found that NF-κB activity and IκBα protein degradation were inhibited by berberine sulfate. In addition, NLRP12 serves as a negative regulator of inflammation and osteoclast formation via inhibition of IκB-α degradation and downregulation of the NF-κB pathway [62]. Therefore, we investigated the effects of SPN on the NF-κB pathway by detecting NF-κB transcriptional activity and IκBα protein expression. We found that SPN inhibited RANKL-induced NF-κB activity and prevented IκB-α degradation, indicating that SPN suppressed RANKL-induced NF-κB activation. Studies have shown that MAPKs play a key role in the regulation of bone formation and bone homeostasis, especially in the differentiation of osteoblasts and osteoclasts [63]. ERK, JNK and p38 belong to the traditional MAPK family kinases [64]. At the early stage of osteoclast differentiation, RANKL can induce the temporary activation of the MAPK signaling pathway. Moreover, downregulation of triggering receptors expressed on myeloid cells 2 (TREM-2) expression can weaken the activation of the RANKL-induced calmodulin-dependent protein kinases (CaMKs)-MEK-ERK signaling pathway and reduce the expression of NFATc1 [65]. Therefore, we investigated the effects of SPN on the MAPK pathway by detecting p-ERK, ERK, p-p38, p38, p-JNK, and JNK expressions. We found that SPN significantly attenuated p-ERK/ERK, p-p38/p38, and p-JNK/JNK expressions. In general, we conclude that SPN inhibits the activation of RANKL-mediated NF-κB and MAPK signaling pathways at the cellular level in the early stage of osteoclast formation, which inhibits the activation of downstream factors, such as NFATc1 and c-Fos. We further tested the role of SPN in vivo using a diabetic rat model. Consistent with the in vitro results, our in vivo study showed that SPN prevented bone loss and osteoclast formation. Moreover, we analyzed the role of SPN on diabetes. Interestingly, SPN decreased the serum glucose, HbA1c, LDL-C, TC and TG levels and effectively improved the insulin and HDL-C levels. These results suggested that SPN had a protective role in DM. In addition, a previous study reported that SPN could regulate blood sugar control and glucose tolerance in gestational diabetic rats [24]. These findings suggest that SPN suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis. However, this study still has some limitations. For example, in the present study, only a part of cancellous bone in femur was extracted to perform 3D reconstructions and H&E staining. In addition, further work is required to determine whether SPN inhibits osteoclast formation and bone loss through the NF-κB and MAPK pathways.
In summary, we found that SPN has an inhibitory effect on the osteoclast formation of BMMs, suggesting that SPN may have antiosteoclast and anti-bone resorption activities. We further elucidated the molecular mechanism of the SPN effects, including inhibition of NF-κB activation and the levels of downstream factors c-Fos and NFATc1, and inhibition of JNK and p38 phosphorylation. Therefore, we believe that SPN may help treat osteoclast-related bone diseases, although further studies are needed to determine the appropriate dose and treatment strategy. SPN may also be used as a potential traditional Chinese medicine to prevent and treat diabetes-induced osteoporosis disease. In the era of precision medicine, accurate targeting and appropriate drug concentrations are the keys for using natural compounds to treat osteoclast-related osteolytic diseases in the future.

Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica Sinica online.